This is the Huber Lab RNA SIP protocol that was co-written by Caroline Fortunato, Amy Smith, Sabrina Elkassas, and Olivia Ahern. It was last updated 27 September 2022 by Olivia Ahern. Another version of this protocol, using the old CsTFA formulation (liquid) can be found on protocols.io.
This protocol is to extract RNA and fractionate from 20% 13C-labelled cultures from Siders Pond into 22 fractions. The experiment that used this protocol can be found at Olivia Ahern’s github page.
Items used in protocol:
Before beginning the extraction protocol:
Cell Lysis
Precipitation of total nucleic acids
Removal of contaminating DNA from total nucleic acids preparation
Materials:
Prior to Start:
At least 1 hour before you are ready to read, turn on the computer and open the software for the plate reader (Software: SoftMaxPro). THEN turn on the plate reader. The software will not find the plate reader unless you follow in this order; click on corner icon (SpectraMax), select instrument to connect. During warm-up of plate reader, follow the protocol listed below.
Plan your plate. You will need one well for each of 7 standards (add the number of l equal to the ng/mL; 50 ng/mL = 50 L) and one well for each sample. You can reduce or add standards if you think your nucleic acid falls outside of the standard range. Do not use the edge rows/columns (plate reader may not be accurate here at all edge wells). Also, run duplicates of unfractionated RNA.
Make 1:2000 Ribogreen mixture from aliquot of Ribogreen reagent and 1X TE. E.g. If you need a total of 2 mL of 1:2000 Ribogreen, mix 1 L Ribogreen with 1999 L 1X TE. Protect from light.
Set up plate:
| Well | Volume 1X TE (μL) | Volume of RNA (μL) | RNA Concentration (ng/μL) |
|---|---|---|---|
| B2 | 49 | 1* | 100 |
| C2 | 0 | 50 | 50 |
| D2 | 25 | 25 | 25 |
| E2 | 40 | 10 | 10 |
| F2 | 45 | 5 | 5 |
| G2 | 49 | 1 | 1 |
| B3 | 50 | 0 | 0 |
*use 1:10 – 10 g/ml RNA concentration (ng/mL) Fill rest with nucleic acid 49 1 Determine by standard curve
Settings:
Read Mode: FL
Wavelength: 480-520 nm
Read Type: Well Scan (will take the average of 5 readings in each well)
Plate type: 96 well standard opaque
Read Area: highlight and assign standards, blanks, and sample wells (avoid using outer wells)
Template Editor:
Indicate wells containing standards and enter their concentration
Standard curve will require you to input the concentration but it is in mg/ml, this is OK. Fill in the above values from your standard curve
Also denote the plate blank
Indicate wells containing samples – click assign each time you highlight a well.
Press Read
Calculations
Using the ribogreen measurements, calculate how many μL of RNA is needed from each sample for a total of 500-750 ng (I use 500 ng). You may need to make 10X, 100X, or 1000X dilutions of your RNA in 1X TE in order to measure them properly. I always do a freeze thaw after my RNA extraction to make sure my RNA is properly hydrated.
This modification comes from the Buckley lab
Items needed:
Recipe from Dunford and Neufeld, 2010
Combine
The final solution is 0.1 M Tris, 0.1 M KCl and 1 mM EDTA.
The day before you setup a spin, hydrate your 50 g powdered CsTFA with 13 mL of GSS. Shake vigorously and allow to come to room temperature for several hours to overnight (I do overnight). Measure the RI of the GSS you hydrate the CsTFA with (after the calibration). GSS can increase in density due to evaporation over time.
Note: CsTFA is extremely hydroscopic (i.e. loves water) and will become crusty if you leave the powder out after opening the bottle (i.e. absorbs water through humidity). This step ensures consistency between SIP spins over time.
Protocol is a derivation of the Huber lab protocol, Lueders 2010, Buckley Lab RNA SIP Protocol, and Roey Angel’s protocol
Protocol
Note: when the cycle is finished, it will not say “end” or “complete;” it will look like you didn’t start it at all. This is why it is important to check on the ultracentrifuge every day that it is running, to make sure it is still running correctly.
Optimal arrangement of tubes in rotor VTi 65.2
Items needed
Protocol
Example Plate
Left. Fractionating into 96-well plates. Right. After fractionating, make sure all of your fractions are level (contain the same amount of CsTFA+RNA).
Before precipitating – if you have an odd number of plates – make a “buddy” plate for the isopropanol and ethanol washes using water. Make sure that it weighs about the same as the plate with CsTFA. Note: CsTFA is heavier than water, so you may need to add like 1.1-1.3X more water to each well to compensate for differences in density.
Ribogreen all fractions to determine concentrations. Using the protocol described on p. 4-5 but with 2 μL template and modified plate setup to allow easier quanitifcation from 96 well plates.
Example ribogreen plate for RNA-SIP fractions from 96 well plates. Std = standard.
We use correction factors to compensate for differences in the GSS and formamide between runs. This was not done previously with the liquid CsTFA solution. There are two ways to correct currently. The first is from Lueder’s, which is not a published protocol. The second is from the Buckley lab. At this point and time (Sept 2022) there has been no recent research published on correction factors for RNA SIP (except Buckley’s).
Use CsTFA standard curve from figure 2B (below) Lueders 2010 to convert RIs into buoyant density and apply the correction factor.
Relationship between RI and Buoyant Density from Leuders
Correction factor:
(density of CsTFA + Gradient Buffer + formamide: 1.98) - density of CsTFA solution in water adjusted to RI of bought solution.
Therefore, if the RI of your CsTFA + Gradient Buffer + formamide was 1.3783, then the buoyant density of is 1.91549008. Your correction factor would be 0.06450992 (1.98-1.91549008). The formula in excel is: 1893.982POWER(RI_CsTFA+GSS+Form,2)+5230.8018( RI_CsTFA+GSS+Form)-3609.6804).
This is derived from the Buckley Lab SIP Protocol
We calculated the correction factor in step 13 of Gradient Setup. Subtract that value (i.e 0.0051) from all of your RI values from your SIP spin and then calculate using the equation in the link:
RI corrected = RI observed – (RI Buffer - 1.3333)
RI observed = Either the GSS + CsTFA or fractions
RI Buffer = GSS + Formamide (no CSTFA)
Buoyant Density = 163.559 – 262.271*(RI corrected) + 105.281*(RI corrected)^2
Follow using the manufacturer’s instructions with 10 μL reactions and 1 μL of RNA for input.
Master Mix
| Reagent | Initial Conc. | Final Conc. | Volume 1 rxn (μL) | Volume 96+6 rxns (μL) |
|---|---|---|---|---|
| SYBR 2X buffer | 2X | 1X | 5 | 540 |
| dH2O | - | - | 3 | 324 |
| 805R Primer | 10 μM | 0.4 μM | 0.4 | 43.2 |
| 341F Primer | 10 μM | 0.2 μM | 0.2 | 21.6 |
| 50X Rox High | 50X | 1X | 0.2 | 21.6 |
| RT Mix | 50X | 1X | 0.2 | 21.6 |
| Template | - | - | 1 | 108 |
First, normalize your RNA concentrations to the SIP fraction with the highest concentration (Ratio of Maximum Quantity). Graph your 12C control and 13C-labelled SIP fractions with corrected Buoyant Density on the x-axis and normalized Ratio of Maximum Quantity on the y-axis.
library(ggplot2)
library(tidyverse)
qpcr=read.csv(file='/Users/oliviaahern/Documents/R/Exp4/25Aug22/qpcr_6Oct22.csv',
header=T)
# dim(qpcr)
qpcr_sub=subset(qpcr, Buckley > 1.74)
make_bar_relabun <- function(df, selection){
df_out <- df %>%
filter(MC %in% c("MC1",selection))
ggplot(df_out, aes(x = Buckley, y = qPCR_RMAX)) +
scale_fill_manual(values=colls) +
scale_colour_manual(values=colls) +
facet_grid(~TP, scale='free_x', space="free", shrink=TRUE) +
geom_point(aes(colour=MC,
fill = MC), size=1.3) +
geom_line(aes(colour=MC)) +
labs(x="Buoyant Density (g/mL)", y = "Ratio of Maximum Quantity") +
theme_bw() + scale_x_continuous(limits = c(1.76, 1.83)) +
theme(axis.text = element_text(color ='black',size=6, hjust=1,vjust=1),
axis.text.x=element_text(size=6, angle=90),
axis.title = element_text(color='black',face='bold',size=8),
legend.position = "none",
panel.grid=element_blank(),
legend.text = element_text(size=8),
legend.key.size = unit(0.25,'cm'),
strip.text.x = element_text(
size = 10, color = "black", face = "bold"),
strip.background = element_rect(
color="gray80", fill="gray97", size=1, linetype="solid"),
panel.spacing = unit(0.05, "lines")
)
}
colls=c('gray50',"#a44f9a")
m2=make_bar_relabun(qpcr_sub, "MC2") + geom_text(data = data.frame(x = 1.81, y = 1, TP=1.5, label = "MC1 12 Control"),
aes(x = x, y = y, label = label), size = 3,
colour="gray50",fontface='bold') + geom_text(data = data.frame(x = 1.81, y = .90, TP=1.5, label = "MC2 13C-Methanol"),
aes(x = x, y = y, label = label), size = 3,
colour="#a44f9a", fontface='bold')
colls=c('gray50',"#6870c8")
m3=make_bar_relabun(qpcr_sub, "MC3")+ geom_text(data = data.frame(x = 1.81, y = 1, TP=1.5, label = "MC1 12 Control"),
aes(x = x, y = y, label = label), size = 3,
colour="gray50",fontface='bold') + geom_text(data = data.frame(x = 1.81, y = .90, TP=1.5, label = "MC3 13C-Ethanol"),
aes(x = x, y = y, label = label), size = 3,
colour="#6870c8", fontface='bold')
colls=c('gray50',"#56ae6c")
m4=make_bar_relabun(qpcr_sub, "MC4") + geom_text(data = data.frame(x = 1.81, y = 1, TP=1.5, label = "MC1 12 Control"),
aes(x = x, y = y, label = label), size = 3,
colour="gray50",fontface='bold') + geom_text(data = data.frame(x = 1.81, y = .90, TP=1.5, label = "MC4 13C-Acetate"),
aes(x = x, y = y, label = label), size = 3,
colour="#56ae6c", fontface='bold')
colls=c('gray50',"#af953c")
m5=make_bar_relabun(qpcr_sub, "MC5") + geom_text(data = data.frame(x = 1.81, y = 1, TP=1.5, label = "MC1 12 Control"),
aes(x = x, y = y, label = label), size = 3,
colour="gray50",fontface='bold') + geom_text(data = data.frame(x = 1.81, y = .90, TP=1.5, label = "MC5 13C-Glucose"),
aes(x = x, y = y, label = label), size = 3,
colour="#af953c", fontface='bold')
colls=c('gray50',"#ba4a4f")
m6=make_bar_relabun(qpcr_sub, "MC6") + geom_text(data = data.frame(x = 1.81, y = 1, TP=1.5, label = "MC1 12 Control"),
aes(x = x, y = y, label = label), size = 3,
colour="gray50",fontface='bold') + geom_text(data = data.frame(x = 1.81, y = .90, TP=1.5, label = "MC6 13C-Xylose"),
aes(x = x, y = y, label = label), size = 3,
colour="#ba4a4f", fontface='bold')
library(cowplot)
plot_grid(m2,m3,m4,m5,m6,aligh='hv',ncol=1)
Above are buoyant density curves over time from a batch experiment using 20% 13C-labelled substrates vs. the control. Note how different substrates show different buoyant density curves over time.
Determine how many RNA SIP fractions you want to send for 16S and 18S amplicon sequencing and then proceed to the next step.
This protocol is from Biorad’s iScript Select cDNA synthesis
This is for 20 μL reactions. But you can use 10 μL reactions if just using the cDNA product for one amplicon PCR.
Protocol 20 μL Reactions
In PCR hood, make up master mix over ice and dispense 7 μL into each PCR tube. Add nuclease free water in hood and then add template out of hood.
We normalize each of our reactions:
| Component | Vol/Rxn | Vol/13 Rxns |
|---|---|---|
| 5x iScript Reaction Mix | 4 | 52 |
| iScript Reverse Transcriptase | 1 | 13 |
| Random Primers | 2 | 26 |
| Nuclease-free water | Variable | Variable |
| RNA template (100 fg - 1 ug total) | Variable | Variable |
Place into thermocycler and run program under Olivia A > iScript cDNA 1708897.
Thermocylcer Program
Priming 5 min at 25°C
Reverse Transcription 30 min at 42°C
RT inactivation 5 min at 85°C
Optional Hold at 4°C
Read protocol from above.
We are sending our cDNA libraries to IMR for 16S and 18S sequencing.
| Item Description | Cat. No. |
|---|---|
| KAPA SYBR FAST One Step qRT-PCR Universal 1,000 20 uL rxns | KK4652 |
| MasterPure™ Complete DNA and RNA Purification Kit | MC85200 |
| Eppendorf 500 μL 96 deep well plates | 9510131801 |
| Eppendorf 96 deep well plate sealing mat | 0030127978 |
| Millipore Express 0.2 μM Filters 25 mm | GPWP02500 |
| Cesium Triflouroacetate 50g | AA4463318 |
| iScript Select cDNA Synthesis Kit | 1709987 |
| BioRad “B” PCR Plate Sealing Film | MSB1001 |
| BD Precision Glide Needles, 23 gauge | 14-826A |
| USA Scientific PCR Cooler Tube Rack | 4051-0509 |
| Corning Sterile Reagent Reservoirs | 4870 |
| GoTaq DNA Polymerase | M3008 |
| ThermoScientific Tris-HCL, 1M Solution pH 8.0, Ultrapure/molecular grade | J22638-AE |
| Invitrogen 0.5 M EDTA pH 8.0 | AM9260G |
| DEPC-Treated Water | AM9906 |
| Beckman optiseal polypropylene centrifuge tubes, 4.9 mL | 362185 |
| ThermoScientific Potassium Chloride, 99% Molecular Grade | J64189.36 |
| Fisher Tris-EDTA (10X Solution), pH 7.4 | BP2477-100 |
| Axygen 1.7mL low retention microcentrifuge tubes | MCT-175-C-S |
| Axygen 0.2ul low retention, Maximum Recovery, PCR tubes | PCR-02-L-C |
| BioRad Multiplate PCR Plates, 96-well clear | MLL9601 |
| Costar 96-well Black Flat bottom Polystrene, 25 per pack | 3915 |
| SuperScript® III First-Strand Synthesis System | 18080-051 |
| NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module | E6111S |
| RNeasy MinElute Cleanup Kit; 50 rxns | 74204 |
| Ovation Complete Prokaryotic RNA-Seq DR Multiplex System 1-8 (32 rxn) | 0326-32 |
| Agilent High Sensitivity DNA Kit | 5067-4626 |
| Agilent DNA 1000 kit | 5067-1504 |
| Saga Sciences 1.5% agarose cassettes | BDF1510 |
| Saga Sciences gNDA for 1.5% internal standard gel cassettes | CIS1504 |
Sourced isotopes from Cambridge Isotope Laboratories. Sourced primers from New England Biolabs (100 μM, hydrated).